Blood glucose concentrations serve as an important marker for diabetes mellitus. Heretofore, enzymatic methods using glucose oxidase (GOD), glucose-6-phosphate dehydrogenase (G6PDH), glucose dehydrogenase (PQQGDH) whose coenzyme is pyrroloquinoline quinone, or the like have been used for measuring glucose concentrations. GOD, however, requires oxygen as an electron acceptor. Its measurement value is therefore disadvantageously influenced by the level of dissolved oxygen in a test sample. G6PDH inevitably involves adding its coenzyme NAD(P) to the reaction system and disadvantageously complicates the detection system. Furthermore, PQQGDH has unfavorable high activity against maltose due to its low glucose selectivity, though this enzyme advantageously has high oxidizing activity against glucose, which eliminates the need of oxygen as an electron acceptor. Thus, there has been a demand for a novel enzyme that can be used as a sensing element for glucose sensors. In addition, the enzyme is desired to have such low reactivity with xylose that blood sugar levels can be accurately measured even during a xylose absorption test.
Fungi have been classically known to have glucose dehydrogenase (e.g., Biochim biophys Acta. 139 (2), p. 265-276, 1967). Aspergillus- or Penicillium-derived glucose dehydrogenase and glucose concentration measurement using this enzyme are disclosed in, for example, the following patent literatures (Japanese Patent Laid-Open No. 2007-289148, Japanese Patent Laid-Open No. 2008-178380, Japanese Patent Laid-Open No. 2008-035748, Japanese Patent Laid-Open No. 2008-035747, WO2007/11610, WO2004/058958, WO2006/101239, and WO2007/139013). Most of fungus-derived enzymes, however, are glycoproteins and thus require glycosylation for expressing their functions. Particularly, extracellularly secreted enzymes, such as glucose oxidase, are highly glycosylated. These fungus-derived glycoproteins are therefore very difficult to produce recombinantly using E. coli. Since fungus-derived glucose dehydrogenase is also an extracellularly secreted protein, this fungus-derived glucose dehydrogenase is difficult to express recombinantly in E. coli. Even if this enzyme can be expressed in such a manner, the enzyme has unfavorable low yields per unit volume of cultures due to its low productivity (e.g., Biotechnol. Lett. Volume 33, Number 11, p. 2255-2263, 2011).
All references cited herein are described below. The contents described in these literatures are incorporated herein by reference in their entirety.